Cintia Hiromi Okino

Estimates of repeatability and correlations of hemoparasites infection levels for cattle reared in endemic areas for Rhipicephalus microplus.

Rhipicephalus microplus is a vector of cattle tick fever, a disease caused by the protozoans Babesia bovisand B. bigemina, and also anaplasmosis, produced by the Rickettsiales Anaplasma marginale. These tick-borne pathogens cause considerable losses to Brazilian livestock breeders and represent an obstacle to the expanded use of taurine breeds due to their higher sensitivity to ticks and hemoparasites compared to zebu breeds. Differences in the susceptibility to hemoparasites were also verified within breeds, suggesting that may be possible to select a most resistant phenotype. Therefore, repeatability of R. microplus counts and copy number of hemoparasites DNA were estimated, along with correlations between themselves, aiming to verify if those measures can be used as parameters to classify animals according to their parasite resistance degrees. Forty-two Canchim females kept on pastures naturally infested by ticks were evaluated for the level of infestation by R. microplus and infection by B. bovis, B. bigemina, and A. marginale. Twenty-four evaluations were performed once a month, for adult female ticks counts and blood samplings. The experimental period was divided into four phases, according to the animals age range: Phase 1: 8 to 13 months (collections 1 to 6); phase 2: 14 to 19 months (collections 7 to 12); phase 3: 20 to 25 months (collections 13 to 18), and phase 4: 26 to 31 months (collections 19 to 24). Blood samples were submitted to absolute quantification of hemoparasites DNA sequences using qPCR. The hemoparasite and tick counts data were transformed for normalization and were analyzed using mixed models. Among three species of hemoparasites studied, A. marginale presented the highest level of infection. During phase 3, B. bigemina presented higher infection levels (pu202f<u202f0.05) compared to B. bovis, whereas no differences were observed in other phases. Estimated repeatabilities for parasite infection levels varied from low to moderate during our experiment. There were low correlations between tick counts and parasite infection levels, and between parasite infection levels from different species by themselves. Based on these results, under conditions of the present study, we suggest that it is possible to identify animals presenting a most resistant phenotype against infection by both hemoparasites and ticks. Moreover, the animal age may be an important factor related to resistance against these pathogens. The data obtained shed more light on the resistance to hemoparasites studied.

Inactivated infectious bronchitis virus vaccine encapsulated in chitosan nanoparticles induces mucosal immune responses and effective protection against challenge

Avian infectious bronchitis virus (IBV) is one of the most important viral diseases of poultry. The mucosa of upper respiratory tract, specially the trachea, is the primary replication site for this virus. However, conventional inactivate IBV vaccines usually elicit reduced mucosal immune responses and local protection. Thus, an inactivated IBV vaccine containing BR-I genotype strain encapsulated in chitosan nanoparticles (IBV-CS) was produced by ionic gelation method to be administered by oculo-nasal route to chickens. IBV-CS vaccine administered alone resulted in markedly mucosal immune responses, characterized by high levels of anti-IBV IgA isotype antibodies and IFNγ gene expression at 1dpi. The association of live attenuated Massachusetts IBV and IBV-CS vaccine also induced strong mucosal immune responses, though a switch from IgA isotype to IgG was observed, and IFNγ gene expression peak was late (at 5u202fdpi). Efficacy of IBV-CS was evaluated by tracheal ciliostasis analysis, histopathology examination, and viral load determination in the trachea and kidney. The results indicated that IBV-CS vaccine administered alone or associated with a live attenuated heterologous vaccine induced both humoral and cell-mediated immune responses at the primary site of viral replication, and provided an effective protection against IBV infection at local (trachea) and systemic (kidney) sites.

Impact of treatments for recycled broiler litter on the viability and infectivity of microorganisms

The microbiological risk of recycled litter depends on the efficacy of the management system applied to inactivate residual microorganisms and preserve the health of the successive broiler flock. This study aimed to assess the viability and infectivity of the Newcastle Disease Virus (NDV), Infectious Bursal Disease Virus (IBDV) and Salmonella Heidelberg in recycled litter exposed to different treatments. The litter was contaminated with microorganisms and submitted to the treatments (T): T1: shallow fermentation; T2: quicklime (calcium oxide); T3: shallow fermentation followed by addition of quicklime; T4: no treatment. Sentinel chicks housed on the treated litter showed that T1 and T3 inactivated residual IBDV. Analysis of the litter subjected to T1 also showed reduced levels of total enterobacteria. T2 was not able to reduce the microorganisms assessed and its association with T1 (T3) failed to enhance the effect of the treatment. NDV did not survive in the broiler litter, regardless of the treatment applied, and it was also not detected in the sentinel chicks. S. Heidelberg remained viable in the litter submitted to all studied treatments, being isolated from the sentinel chicks of all the experimental groups. The antimicrobial activity of T1 and T3 was associated to higher ammonia contents in the broiler litter. The results indicate that the shallow fermentation treatment is efficient for controlling residual IBDV and total enterobacteria in the recycled litter.

Identification of appropriate reference genes for local immune-related studies in Morada Nova sheep infected with Haemonchus contortus

Due to the great economic impact of Haemonchus contortus on sheep farming, there is an increasing number of studies addressing host resistance against this nematode, including identification of directly related immune mechanisms. In this context, relative gene expression by RT-qPCR have been largely used, due to its rapidity, high sensitivity, specificity, and reproducibility. Although, appropriate reference gene selection is crucial for accurate interpretation of results. In this study, five reference genes (GAPDH, G6PDH, YWHAZ, ACTB, and B2M) were tested for expression stability in abomasum (fundic and pyloric regions) and abomasal lymph nodes of Morada Nova sheep classified as resistant (nu2009=u20095) or susceptible (nu2009=u20095) to H. contortus infection in a flock of 151 animals. GAPDH combined with YWHAZ were selected as reference genes for abomasal fundic region and abomasal lymph nodes, whereas YWHAZ was the most stable gene for abomasal pyloric region. These genes presented the lowest intra- and inter-group variations and, consequently, highest stability. In contrast, expression of G6PDH was the least stable in all tissues. The impact of reference gene selection was demonstrated by relative quantification of a pro-inflammatory cytokine (TNFα) in abomasal fundic region. Significant differences in TNFα expression levels between resistant and susceptible groups were only observed when the most stable genes (GAPDH combined with YWHAZ) or GAPDH were used as reference genes, whereas no significant differences were observed when other tested reference genes were used. It was demonstrated that normalization of expression data using inappropriate reference genes may significantly influence interpretation results.

Comparative evaluation of DNA extraction kit, matrix sample and qPCR assays for bovine babesiosis monitoring

Bovine babesiosis caused by protozoan parasites Babesia bovis and B. bigemina is one of the most important causes of losses for the livestock industry in tropical and subtropical regions of the world. Therefore, highly sensitive and specific tools for these hemoparasites detection and monitoring are required, especially in carrier animals, in which low parasite levels were usually present. In this context, qPCR assays have been successfully and fairly used in last years. Aiming to improve the performance of Babesia levels monitoring by qPCR, some of main aspects of this methodology that may influence results were tested: DNA extraction kits, whole blood EDTA pre-treatment, blood source (tip of tail or jugular vein), erythrocytes isolation, FTA card interference and qPCR system of detection. Under our experimental conditions, both EDTA pre-treatment and FTA card application have no influence on the sensitivity of detection, and two DNA extraction kits provided higher sensitivity compared to others. As expected, blood samples collected from the tip of tail vessels presented higher levels of B. bovis DNA compared to those obtained from the jugular vein, and erythrocytes processed isolated has also improved the sensitivity compared to whole blood. Moreover, both qPCR assays here developed using hydrolysis probes for B. bovis and B. bigemina detection, presented enhanced reproducibility compared to qPCR assays using intercalating dye system. Even, qPCR for B. bigemina using hydrolysis probe here developed presented higher sensitivity compared to intercalating dye system. This study has contributed to the improvement of molecular diagnosis of bovine babesiosis, which may improve epidemiological studies related to these pathogens.

Rapid detection and differentiation of avian infectious bronchitis virus: an application of Mass genotype by melting temperature analysis in RT-qPCR using SYBR Green I

A method based on Melting Temperature analysis of Hypervariable regions (HVR) of S1 gene within a RT-qPCR was developed to detect different genotypes of avian infectious bronchitis virus (IBV) and identify the Mass genotype. The method was able to rapidly identify the Mass genotype among IBV field isolates, vaccine attenuated strains and reference M41 strain in allantoic liquid and also directly in tissues. The RT-qPCR developed detected the virus in both tracheal and pulmonary samples from M41-infected or H120-infected birds, in a larger post-infection period compared to detection by standard method of virus isolation. RT-qPCR method tested provided a sensitivity and rapid approach for screening on IBV detection and Mass genotyping from IBV isolates.

qPCR estimates of Babesia bovis and Babesia bigemina infection levels in beef cattle and Rhipicephalus microplus larvae

Babesia spp. are tick-transmitted intraerythrocytic apicomplexan parasites that infect wild and domestic animals. Babesia bovis and B. bigemina are endemic and responsible for enormous economic losses to the livestock industry in most of the Brazilian territory, wherein the tick Rhipicephalus microplus is the unique vector. Better understanding of epidemiology and parasite–host interactions may improve the tools for disease control and genetic management for selection of resistant animals. This study aimed to detect, quantify and measure the correlation between B. bigemina and B. bovis infection levels in bovine blood and into tick, by absolute quantification of hemoparasite DNA using qPCR. Blood bovine samples and larvae pools from 10 engorged R. microplus females were collected from each Canchim heifers (5/8 Charolaisu2009+u20093/8 zebu, nu2009=u200936). All evaluated samples were positive for both Babesia species tested. Correlations of B. bovis and B. bigemina levels between cattle and tick host were 0.58 and 0.66, respectively. These high positive correlation coefficients indicate that parasitemia load in the bovine may be dependent on or may determine the parasitemia load in the ticks.